Various methods for producing vaccines and other biologics in cell cultures have been pre-described. If continuous cell lines are used for the production, there is the risk that residual DNA of the cell line could be oncogenic. It is therefore required to destroy and remove residual DNA from therapeutic proteins of interests. For viral vaccines the FDA currently recommends a DNA amount of less than 10 ng/dose and a fragment size of less than 200 base pairs (Guidance of Industry. Characterization and Qualification of Cell Substrates and other Biological Materials Used in the Production of Viral Vaccines for Infections Disease Indications. FDA/CBER February 2010; downloadable under: fda.gov/downloads/biologicsbloodvaccines/guidancecomplianceregulatoryinformation/guidances/vaccines/ucm202439.pdf).
Several methods for removing residual DNA from cell culture derived vaccines have been described. U.S. Pat. No. 5,948,410 describes a method for producing flu vaccines derived from cell culture in which a DNAse treatment is combined with a splitting step using CTAB. WO 2007/052163 describes a method for producing flu vaccines derived from cell culture in which beta propiolactone (BPL) is used to inactive the virus and to degrade the residual DNA. Afterwards, the virus is split, e.g., by treatment with CTAB. The fragmented DNA is then removed from the virus preparation. Nevertheless, there is still the need to further improve removal of residual cellular DNA from influenza virus preparation or from other products of interest produced in continuous cell lines.
The use of caprylic acid in combination with ion exchange chromatography for the removal of cellular DNA from antibodies produced in cell culture has been disclosed in US 2012-0101262. However, US 2012-0101262 requires the use of caprylic acid under conditions that induce precipitation of residual DNA and contaminating proteins (in particular at low pH). Afterwards, the precipitate and the protein of interest can be separated, and the latter is further purified via ion exchange chromatography.
Various other methods of removing impurities and aggregates derived from cell cultures using caprylic acid or caprylate salts have been described in the art. Steinbuch (Steinbuch, M. et al. Arch. Biochem. Biophys. 134:279-94 (1969) describes recovering IgG from human plasma by caprylate precipitation of nonenveloped and enveloped viruses therein. U.S. Pat. No. 7,553,938 describes purification of antibodies from a starting solution by adding caprylate or heptanoate ions at pH 4.6 to about 4.95 and filtering the solution through at least one anion exchange resin. U.S. Pat. No. 5,886,154 describes a process for purification of antibodies from human plasma involving suspension of antibodies at pH 3.8 to 4.5 followed by addition of caprylic acid at pH 5.0 to 5.2 to precipitate contaminating proteins and lipids while the antibodies remain in solution. The use of caprylic acid is employed in antibody purification because short fatty acids form insoluble complexes with alpha and beta globulins and at acidic pH whereas the gamma globulins are not as readily precipitated (Chanutin et. al., 1960). Thus the gamma globulin can easily be separated. Yet, none of these disclosures teach or suggest using an anionic detergent to remove residual DNA from viral proteins under conditions which prevent precipitation as taught herein.